Although oxygen is essential for aerobic life, reactive byproducts of respiration such as superoxide anion(02-)can be toxic to biomolecules, and have been associated with numerous human disease states. One mechanism by which organisms are thought to combat oxygen toxicity involves the 02- scavenging activity of superoxide dismutase (SOD). Remarkably, cells lacking SOD are not only oxygen-sensitive, but are unable to biosynthesize certain amino acids, suggesting multiple roles for this enzyme in cellular metabolism. The research presented here represents a novel approach to understanding the complex function(s) of SOD in cellular metabolism and exploits the combined technologies of yeast genetics and biochemistry. Our laboratory has recently isolated S. cerevisiae suppressors of SOD defect ("SSD" strains), i.e., yeast cells lacking SOD, yet resistant to oxygen toxicity and/or amino acid deprivation. Through accomplishment of the following specific aims, the goal of this study is to characterize the mutation(s) in these SSD strains that bypasses the requirement for SOD: (AIM 1) To understand the biochemical basis for oxygen resistance in the SSD strain: Standard biochemical assays will be performed to monitor production and metabolism of reactive oxygen in the SSD strains. (AIM 2) To characterize genetically the SSD strains. Yeast genetics will be employed to identify strains suitable for future gene cloning experiments and to determine the number of different SSD complementation/allelic groups. These pilot studies will facilitate our long term goals of cloning the SSD gene from these mutants, identifying and understanding the function of the encoded SSD factor(s), and identifying analogous SSD factors in mammalian cells. Conceivably, SSD factors may include proteins involved in reactive oxygen metabolism, or amino acid metabolism enzymes affected by SOD action. The identification of these factors will better define the roles of SOD in cellular metabolism, and should promote an understanding of the adaptive mechanisms necessary to sustain the aerobic lifestyle.